The cell membrane or plasma membrane surrounds the cytoplasm of living cells, physically separating the intracellular components from the extracellular environment. The cell membrane is selectively permeable to ions and organic molecules and controls the movement of substances in and out of the cell. This membrane serves to separate a cell from its surrounding thereby protecting it from changes in the chemical and physical environment, and also permitting regulation of the entry/exit of molecules into and out of the cell. It is made mostly from a bilayer (double layer) of phospholipid molecules, only very few molecules can pass directly through the lipid bilayer to get from one side of the membrane to the other. Embedded within this membrane are a variety of protein molecules that act as channels and pumps for moving vital molecules into and out of the cell. Therefore, it is said that the cell is selective permeable, able to regulate what enters and exits the cell, thus facilitating the transport of materials needed for survival.
Whilst vital biologically, these membranes often pose a difficulty for the delivery of many therapeutics whose efficacy depends upon such molecules being able to travel through the aqueous environment in the body and subsequently across the hydrophobic barrier of cellular membranes.
Cell-permeable or Cell-Penetrating peptides (CPPs) (also known as a protein transduction domain or membrane translocation sequence) are used to overcome the impermeability of the plasma membrane. Typically less than 30 amino acid residues in length, CPPs can traverse the membranes of a cell and access the cell interior, for this reason they have been exploited in a range of living systems to internalize molecules (generically termed ‘cargo’). Hundreds of different CPP sequences have now been described and all have a universal capacity to breach biological membranes and enter cells, either alone or when associated with cargo. The function of the CPPs is typically to deliver cargo into cells, a process that commonly occurs through endocytosis with the cargo being delivered via the endosomes of living mammalian cells. Coupling of CPPs to proteins, oligonucleotides, peptide nucleic acids, and other pharmacologically active compounds thus provides a promising strategy for cellular delivery of otherwise membrane-impermeable molecules.
Common applications of CPPs include the delivery of nucleic acid-based macromolecules such as siRNA, antisense oligonucleotide, DNA, and plasmids; all have been realized as promising biological and pharmacological therapeutics in the regulation of gene expression. Recently, it has been reported, using several methods, that CPPs can be used as vehicles to deliver biologically active, full-length proteins, such as horseradish peroxidase, RNase A and even CPP-crosslinked Fab fragments, into living cells.
Broadly speaking, CPPs are generally classified into three groups, but all share the ability to translocate the plasma membrane and facilitate the delivery of various molecular cargoes to the cytoplasm or even to an organelle:                i. short sequences of amino acids with a high density of basic (+) charge, commonly a string of Lysine or Arginine residues e.g. octarginine;        ii. Viral peptides, of which the trans-activating transcriptional activator (TAT) sequence from HIV is the most studied; or        iii. The Antennapedia peptide, and derivatives thereof, a key transcription factor discovered within the Antennapedia protein in the 1990s involved in the development of Drosophila.         
Additionally, or alternatively, CPPs may be classified based upon their peptide sequences and binding properties to lipids: primary amphipathic; secondary amphipathic; and non-amphipathic CPPs.
Mechanistically, the ability of CPPs to translocate a membrane is an on-going area of research. It is thought that the mechanism of action and can vary from one CPP to the next, with some CPPs employing more than one mechanism. Generally, it is thought CPPs enter via: 1) direct penetration of the membrane; 2) endocytosis-mediated entry; or 3) translocation through the formation of a transitory structure.
Direct penetration has recently been proposed to involve strong interactions between cell-penetrating peptides and the phosphate groups on both sides of the lipid bilayer, the insertion of charged side-chains that nucleate the formation of a transient pore, followed by the translocation of cell-penetrating peptides by diffusing on the pore surface.
Endocytosis is the process of cellular ingestion by which the plasma membrane folds inward to bring substances into the cell, but is largely thought to be energy dependent.
In contrast, the third class of CPPs have the property of being internalized by cells through a mechanism that is independent of classic endocytosis. Although the physics of this mechanism is more poorly understood, unlike the other classes, peptides of class 3 can transverse membranes and require no biological surface receptor or cell-derived ATP energy to do so.
In addition to Antennapedia peptide sequence (pAnt), an example of this class 3 includes the synthetically derived PENETRATIN™ CPP, a 16 amino acid peptide derived from the DNA binding domain of the Antennapedia homeoprotein, which is one of the most commonly used CPPs.
Although the class 3 Antennapedia peptide sequence (pAnt) is found within the genome of creatures as diverse as Man, Mouse, Fly and the simple earth worm, there is no Antennapedia sequence in the genome of the eukaryotic Dictyostelium amoeba.
We herein disclose the identification of two novel class 3 CPPs from the genome of the social amoeba Dictyostelium discoideum, herein termed CUPID A and CUPID B in reference to their origin (Cellular Permeating peptides In Dictyostelium). The incorporation of these sequences into larger polypeptides or protein sequences, using recombinant methods, provides products that can permeate cells whilst retaining their polypeptide or protein functionality. Advantageously, these CUPID peptides have been shown to result in superior transport of cargo into cells when compared to other tested CPPs, such as the commonly used PENETRATIN™ CPP, and therefore the CUPID peptides offer improved transport performance for the intracellular delivery of otherwise cell impermeable molecules.